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Stuckey, Jeff on
URL: http://confocal-microscopy-list.275.s1.nabble.com/mEos4-photoconversion-tp7583802p7583804.html
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Hi Jordan,
I'll be around Bill's lab next week a few times collecting data. In line with Sarah's suggestion, I can show you how to use the transmitted light along with DIC in order to find your cells. Jimmy and I talked about this last night after you left. It's a new capability where the system can do DIC imaging through the confocal scanner.
Best
Jeff Stuckey
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From: Confocal Microscopy List [mailto:
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Sent: Friday, May 29, 2015 8:35 AM
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Subject: mEos4 photoconversion
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Hey I hope somebody can help me out. I've started working with a fusion protein of mEos4a and the photoconversion has been working poorly. I'm using a swept field confocal and Prairie View software.
I am using a 488 laser at 20% power and 561 at 35% power for visualizing the protein in live cells. I notice that the green form of mEos bleaches quite quickly even as I'm identifying cells of interest to image. In addition, there tends to be quite a bit of red signal already detectable in these cells without photoconversion by a 405 laser.
I am able to get ~500% increase in red signal and maybe a 50% decrease in green signal upon 405 conversion (but still far from complete and it was difficult to find cells with minimal red signal). I converted using 20 fast repetitions of a relatively low powered 405 (~10%) at a single point (0.500micron diameter).
Does anyone have any advice for best visualization laser and photoconversion laser settings? I'm looking to use mEos4 for FRAP/FLAP. Is mEos sensitive to ambient light
Best,
Jordan